Higher plants contain a family of actin genes made up of many ancient and diverse classes. It is proposed that the ancient classes of plant actin genes have been preserved throughout vascular plant evolution because they have unique patterns of gene regulation and/or encode actin proteins with unique functions. A major goal of this research is to define the differential expression of these classes with respect to cell, tissue and organ type, and development. An initial physical characterization of several actin genes and diverse protein isovariants in Arabidopsis suggests it is the ideal plant in which to pursue this goal and that results will be representative of most angiosperms. One set of objectives focuses on delineating the patterns of expression for the different gene family members in Arabidopsis. To accomplish this an mRNA phenotype will be determined for variety of cell types and tissues beginning with those in root. Polymerase chain reaction (PCR) mediated sequencing of transcripts from a single cell type or tissue will unambiguously define the mRNA phenotypes. Internal controls will be used to quantify expression. A second goal focus on defining the roles for actins in different cells and tissues and in plant development. Specific objectives include making two types of actin mutants. Plants heterozygous for T-DNA insertions in actin genes will be isolated from a large population of T-DNA insertion mutants using a PCR screening method. An alternative approach makes use of mosaic plant actin mutants to preserve the viability of the parent organism. A dormant indicator beta-glucuronidase (GUS) gene fused to an antisense actin RNA will be activated by the loss of a Ds transposon in the transcript leader. This approach has the additional advantage of expressing the mutant phenotype in a developmental pattern. Plants, organs and cell lineages showing mutant phenotype or expressing GUS will be characterized cytochemically and biochemically for actin expression. An ultimate goal is to correlate the diversification of actin gene family members with the origin of cell types and tissues in vascular plants and with the numerous functions proposed for plant actins.